The measurement of aqueous solubility in a high-throughput screening environment plays an important role in the selection of the most promising drug candidate molecules in pharmaceutical research and development. The invention described involves a method, a simple, robust, high-throughput screen, that is applicable for the determination of the equilibrium solubility of sparingly soluble compounds and that may be used in pharmaceutical, biotechnology, and related industries. An analytical device has been designed to implement this solubility measurement technique.
The 1990 treatise by Grant and Higuchi [D. J. W. Grant, T. Higuchi, Solubility Behavior of Organic Compounds, John Wiley and Sons: New York, 1990] comprehensively covers the known art. Many protocols have been described in the literature [S. Venkatesh, J. Li, Y. Xu, R. Vishnuvajjala, B. D. Anderson, i Pharm. Res. 1996, 13, 1453-1459; A. Avdeef, i Pharm. Pharmacol. Commun. 1998, 4, 165-178; A. Avdeef, C. M. Berger, C. Brownell, i Pharm. Res. 2000, 17, 85-89; A. Avdeef in B. Testa, H. van de Waterbeemd, G. Folkers, R. Guy (Eds.), Lipophilicity in Drug Disposition: Practical and Computational Approaches to Molecular Properties Related to Drug Permeation, Absorption, Distribution, Metabolism and Excretion, Univ. Lausanne, 2001, Ch. 22 (in press)] for measuring solubility-pH profiles, using various detection systems.
Classical approaches are based on the so-called saturation shake-flask method, and new rigorous methods are usually validated against it. However, most classical techniques are slow and cannot easily be adapted for the high-throughput needs of modern drug discovery research, one focus of our invention.
At the early stages of research, drug candidate compounds are stored as DMSO solutions, and solubility measurements need to be performed on samples introduced in DMSO, often as 10-30 mM solutions. It is well known that even small quantities of DMSO ( less than 5%) in water can increase the solubility of molecules, and it is a challenge to determine the true aqueous solubility of compounds when DMSO is present, another focus of our invention.
Turbidimetric Ranking Assays
Turbidity detection-based methods, popularized by Lipinski and others [A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Deliv. Rev. 1997, 23, 3-25; P. Q. Charmaine, M. B. Nicholas, A. K. Irwin, Eur. Pharm. Rev. 1998, 3(4); C. D. Bevan, R. S. Lloyd, Anal. Chem. 2000, 72, 1781-1787], in part have met some high-throughput needs of drug discovery research. The methods, although not thermodynamically rigorous, are an attempt to rank molecules according to expected solubilities. Various implementations of the basic method are practiced at several pharmaceutical companies, using custom-built equipment. Detection systems based on 96-well microtitre plate nephelometers have been introduced recently (e.g., LabSystems: Franklin, Mass., USA). The desirable automated solubility analytical device incorporating such a detector requires the operator to develop an appropriate chemistry procedure and to integrate a robotic fluidic system in a customized way. The shortcomings of the turbidity methodology are poor reproducibility (in part due to variability of scattering due to particle size, sometimes erratic scattering due to sedimentation of the sample suspension, and adhesion of suspensions to walls of vessels), the use of excessive amounts of DMSO, and from the view-point of the critical needs of the pharmaceutical industry, the lack of standardization of practice.
HPLC-Based Assays
Several pharmaceutical companies have taken the classical saturation shake-flask method and transferred it onto 96-well plate technology and a robotic liquid dispensing system, in an effort to increase throughput. Analyses are performed with reverse-phase HPLC. Often it is necessary to develop the appropriate chromatographic methods, since in discovery, compounds may not be sufficiently characterized at the early stages. However, generic fast gradient methods may be eliminating the need for method development. In some companies, the DMSO is first eliminated by a freeze-drying procedure, before the aqueous buffers are added, which adds significantly to the assay time and can be problematic with volatile samples. Chromatographic detection systems, although rich in information, being serial, are inherently slow. Data handling is often the rate-limiting step in the operations.
Ideal Solubilityxe2x80x94pH Relationships
For ionizable molecules in dilute solutions (where salt precipitation may be ignored), when the log (logarithm) of the measured solubility is plotted vs. pH, the curve is bilinear, with one segment having a zero slope and the other segment having a unit slope (positive for acids and negative for bases). For example, for an acid such as diclofenac, the solubility is at a minimum for pH less than 3 (where the acid is largely uncharged), stays nearly constant (zero slope segment) as pH is increased, until it exceeds the pKa (3.99), after which the solubility increases by a decade for every increased unit of pH, as more and more of the compound converts from the uncharged state to the more-soluble negatively-charged state (unit slope segment). In a simple aqueous equilibrium system, the pH where the asymptotes of the horizontal and diagonal segments intersect is equal to the pKa of the solute. Under such ideal circumstances, it is possible to determine the pKa and the intrinsic aqueous solubility, So, of the compound from the crossing of the segments [Z. T. Chowhan, J. Pharm. Sci. 1978, 67, 1257-1260; W. H. Streng, H. G. H. Tan, Int. J. Pharm. 1985, 25, 135-145]. Such pKa values can be directly measured by commercially-available devices [C. D. Bevan, A. P. Hill, D. P. Reynolds, Patent Cooperation Treaty, WO 99/13328, Mar. 18, 1999; Sirius Analytical Instruments Ltd., UK].
Complications Which May Thwart the Reliable Measurement of Aqueous Solubility
Certain surface-active compounds when dissolved in water under conditions of saturation form self-associated aggregates or micelles, which can interfere with the determination of the true aqueous solubility and the pKa of the compound [T. J. Roseman, S. H. Yalkowsky, J. Pharm. Sci. 1973, 62, 1680-1685; C. Zhu, W. H. Streng, Int. J. Pharm. 1996, 130, 159-168]. When the compounds are very sparingly soluble in water, excipients are often added to enhance the rate of dissolution. However, the presence of the excipients also can interfere with the determination of the true aqueous solubility. If solubility measurements are done in the presence of simple surfactants [J. Jinno, D. -M. Oh, J. R. Crison, G. L. Amidon, J. Pharm. Sci. 2000, 89, 268-274], bile salts [S. D. Mithani, V. Bakatselou, C. N. TenHoor, J. B. Dressman, Pharm. Res. 1996, 13, 163-167], cyclodextrins [P. Li, S. E. Tabibi, S. H. Yalkowsky, J. Pharm. Sci. 1998, 87, 1535-1537], or ion-pair forming counterions [J. D. Meyer, M. C. Manning, Pharm. Res. 1998, 15, 188-193], extensive considerations need to be applied in attempting to extract the true aqueous solubility from the data, third focus of our invention.
This invention relates to a method for the determination of solubility of a compound and an analytical device for carrying out said method. The basic method involves determining solubility of a compound by measuring the UV spectrum of a reference solution of the compound, under conditions avoiding or suppressing precipitation, and comparing it to the UV spectrum of a saturated sample solution of the compound. Variations of the basic method include: (a) making reference solutions either by dilution of the sample solution to the point where precipitation is avoided, or making reference solutions by adding a water-miscible cosolvent to the sample solution so that precipitation is suppressed, and comparing the UV absorbances of the compound under reference conditions to the compound in a saturated solution, (b) determining the true aqueous solubility from the effect on the pKa that results from dissolving the compound in an aqueous solution containing some DMSO (typically 0.1-5% v/v), and (c) correcting concentrations determined from the UV absorbance values for impurities and other factors that might affect the shape of the sample absorbance curve taken of the saturated solution.
Solubility determinations are illustrated with 15 sparingly-soluble generic, mostly-ionizable drugs (FIG. 1), covering about three orders of magnitude in solubilities, with the lowest value being about 0.1 xcexcg/mL (terfenadine at high pH). The ionizable bases include amiloride, amitriptyline, chlorpromazine, miconazole, nortriptyline, phenazopyridine, propranolol, and terfenadine. The ionizable acid set consists of diclofenac, furosemide, indomethacin, 2-naphthoic acid, and probenecid. Piroxicam was picked as an example of an ampholyte, and griseofulvin as an example of a non-ionizable molecule. The results of the solubility measurements were compared to literature equilibrium solubility-pH values from reliable sources, and to values determined by the pSOL Model 3 (pION) acid-base titrator [A. Avdeef, C. M. Berger, C. Brownell, Pharm . Res. 2000, 17, 85-89].